Process for stabilizing an adjuvant containing vaccine composition

ABSTRACT

The present invention relates to a process for stabilizing an adjuvant containing vaccine composition, an adjuvanted vaccine composition in dry form and in particular a process for stabilizing an influenza vaccine composition, particularly an adjuvanted influenza vaccine composition in dry form.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for stabilizing an adjuvantcontaining vaccine composition, an adjuvanted vaccine composition in dryform and in particular a process for stabilizing an influenza vaccinecomposition, particularly an adjuvanted influenza vaccine composition indry form.

2. Description of Related Art

U.S. Pat. No. 3,655,838 discloses a method of pelletizing analytical orimmunological reagents by freezing droplets of solutions in a freezingmedium, such as liquid nitrogen, and subsequent freeze drying in orderto obtain freeze-dried, reagent containing micro-particles, sphericalbeads or lyospheres. EP 0 081 913 B1 describes a method for producingspherical frozen particles by freezing droplets in an inert liquidfreezing medium with a higher density than the droplets and removing thefrozen particles from the surface of the liquid freezing medium. WO94/25005 discloses the stabilization of gonadotropin in lyospheres, amethod for making such lyospheres and pharmaceutical preparationscomprising the same. EP 0 799 613 (U.S. Pat. No. 5,897,852) discloses avaccine pack that comprises a vaccine container containing one or morefreeze-dried bodies containing the vaccine components, at least one ofwhich being a lyosphere or micro-particle having a diameter of at least0.2 mm. WO 2006/008006 (US 2008/0060213) discloses a process forproducing containers filled with a freeze-dried product wherein dropletsof the product are frozen to form pellets, the pellets are freeze dried,assayed and loaded into the containers. Other techniques for obtainingfrozen particles or pellets are known for application in the foodindustry (e.g. U.S. Pat. No. 5,036,673 or US 2007/0281067 A1).

The freeze drying technology allows improving the stability of a lot ofproducts which can be a vaccine with or without adjuvant. For example EP0 475 409 discloses a method for preserving a buffer and optionally acryoprotectant containing suspension of microscopic biological materialby nebulizing the suspension to microdroplets, freezing the droplets ona rotating cryogenic surface and drying the microdroplets. Preferablythe droplets have a diameter of about less than 200 μm.

The freeze-drying of flu antigens has been studied in the literature anda detailed review is available (Amorij et al. 2008: Development ofstable Influenza vaccine powder formulations: challenges andpossibilities. Pharmaceutical Research, Vol 25, 1256-1273). U.S. Pat.No. 3,674,864 discloses a process for stabilizing influenza virusvaccines essentially by suspending the virus in an aqueous sucrosecontaining solution and freeze-drying the suspension. Also thestabilization of tetanus and diphtheria toxoids has been discussed inthe literature (see e.g. S. P. Schwendeman et al., Proc. Natl. Acad.Sci. USA Vol. 92, pp. 11234-11238, 1995). Very recently, optimizedformulations for lyophilizing tetanus toxoid have been proposed (P.Matejtschuk et al. Biologicals 37 (2009) 1-7).

Usually the freeze drying is a final step in the pharmaceuticalindustry, coming after the filling step in vials, syringes or largercontainers. In this case the dried product has to be rehydrated(synonyms in this document: reconstituted or dissolved) before its use.

Freeze drying in the form of micropellets allows the same stabilizationof the dried vaccine product as for mere freeze-drying alone or itimproves stability for storage. Furthermore, the micropellets technologyoffers several advantages in comparison to the current freeze drying,since it allows e.g.

-   -   blending of the dried products before filling (or by sequential        filling)    -   titer adjustment before filling (which can be used in case of        stock piling)    -   minimizing the interaction between products (there is only        product interaction after rehydration), and    -   improvement of the stability in some cases.

For these reasons the advantage of the micropellets technology allowsseveral approaches for the drying of adjuvanted vaccine:

-   -   The drying of the antigens together with the adjuvant (being in        the same phase): to stabilize the two (antigens and adjuvant)        and to stabilize the interaction between them by trapping them        in a glassy matrix in which all molecular motions and chemical        reactions are greatly hindered. This solid state allows to        maintain throughout storage (even at higher temperature) potency        of the antigen, physical and immunological properties of the        adjuvant and nature and force of the interaction between the        two.    -   The drying of the antigens and the separate drying of the        adjuvant (antigen and adjuvant being in different phases),        followed by blending of the two before the filling or by a        sequential filling. In some cases, stability of the adjuvant        alone can be a problem (chemical stability of emulsions,        physical stability of aluminum gels, liposomes and others . . .        ) at the liquid state for long-term storage at +5° C. or higher        or at lower temperatures. The micropellet technology allows        improving stability of the adjuvanted vaccine by generating        separate micropellets of antigen and adjuvant. The stabilizing        formulation can be optimized independently for each antigens and        the adjuvant. The micropellets of antigens and adjuvants can be        then subsequently filled into the vials or blended before        filling into the vials. The separated solid state allows to        avoid throughout storage (even at higher temperature)        interactions between antigen and adjuvant, to maintain the        potency of the antigen and physical and the immunological        properties of the adjuvant. In such a configuration, the content        of the vial can be more stable than any other configurations        with either one of the antigens or the adjuvants in the liquid        state or when antigens and adjuvant are dried within the same        pellets. Interactions between antigens and adjuvants are this        way standardized as they occur only after rehydration of the dry        combination with a selected diluent which may comprise water for        injection, buffers and stabilizing excipients. Interactions are        therefore to be controlled only during the short period of time        between rehydration and injection of the vaccine.

It is therefore possible to improve the overall stability of the twoproducts can be improved (optimization of the formulation for eachproduct and not a compromise for the two together) and the vaccineitself, adjust the titer of one out of the two after the storage andbefore filling, to facilitate the manufacturing process by separation ofthe two products drying.

SUMMARY OF THE INVENTION

The present invention provides a process for stabilizing an adjuvantcontaining vaccine composition comprising the steps of diluting a liquidbulk composition comprising an antigen or an antigenic preparation by anaqueous solution comprising a stabilizer, subjecting the dilutedcomposition to a process in order to form regular droplets having adiameter of approximately from about 200 μm to about 1500 μm, subjectingthe regular droplets to freezing in a freezing medium to form frozenregular spherical micropellets or particles and drying the frozenregular spherical micropellets or particles to form dry regularspherical micropellets or particles having a diameter from about 200 μmto about 1500 μm.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1. illustrates the general formulation and drying procedure.

FIG. 2. illustrates the process used to generate the dry micropellets,including prilling, freezing, drying and filling.

FIG. 3. illustrates the general formulating, freezing and dryingprocedure comprising an adjuvant.

FIG. 4. illustrates the formulation and drying procedure to produce theantigen containing micropellets.

FIG. 5. illustrates the formulation and drying procedure to produce theadjuvant containing micropellets.

FIG. 6. illustrates the procedure for the combination of the differentmicropellets in order to complete the dry vaccine.

FIG. 7. illustrates the formulation and drying procedure as described inExample 1.

FIG. 8. shows the narrow size distribution of the micropellets obtainedwith pelletizing and drying technology as described in Example 1.

FIG. 9. shows the size distribution results after drying of an aluminumphosphate gel in presence of a carbohydrate and using the micropellettechnology.

FIG. 10. illustrates the formulation and drying procedure as describedin Example 2.

FIG. 11. shows the aluminum oxyhydroxide gel particle size distributionin the formulated bulk before drying and after dissolution of thepellets.

FIG. 12. shows the aluminum oxyhydroxide gel particle size distributionafter thermostability incubation for at least 7 days at 37° C., 45° C.and 55° C.

FIG. 13. illustrates the formulation and drying procedure as describedin Example 3.

FIG. 14. shows a comparison of the size of the emulsion beforerehydration of the micropellets and after dissolution of themicropellets.

FIG. 15. shows the stability of the size of the emulsion afterdissolution of the micropellets and over a one hour storage period atroom temperature.

FIG. 16. illustrates the formulation and drying procedure as describedin Example 4a.

FIG. 17. illustrates the formulation and drying procedure of theformulations containing Diphtheria toxoid and Tetanus toxoid in presenceof aluminum gel as described in Example 5.

FIG. 18. illustrates the formulation and drying procedure of theformulations containing Diphtheria toxoid and Tetanus toxoid withoutaluminum gel as described in Example 5.

FIG. 19. illustrates the formulation and drying procedure of aluminumgel formulation as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

Suitable vaccine compositions are for example compositions comprisingwhole viruses or antigens, such as Influenza, Rotavirus, cytomegalovirus, Hepatitis A and B, whole bacteria or bacterial protein orpolysaccharide antigens (conjugated or non-conjugated), such asHaemophilus influenzae, meningococcal polysaccharides, Tetanus,Diphteria, cellular and acellular pertussis, Botulism, Anthrax or C.Difficile.

The freezing of the droplets can e.g. be achieved in that the dilutedcomposition, i.e. the formulated liquid product, is prilled in order togenerate calibrated droplets which diameter ranges from 200 μm to 1500μm, with a very narrow size distribution. These droplets fall in acryogenic chamber in which the temperature is maintained below −110° C.by a freezing medium, either by direct injection/nebulization of liquidnitrogen or by flowing counter currently a very cold (T°<−110° C.) gassuch as nitrogen, CO₂ or air. The droplets freeze during their fall inorder to form calibrated frozen particles.

Other techniques are known in the art for obtaining calibrated droplets,such as ultrasonic atomization (Sindayihebura D., Dobre M., Bolle L.,1997—Experimental study of thin liquid film ultrasonic atomization.ExHFT'97, Bruxelles.), and, subsequently, calibrated frozen particlesor, as mentioned before being known from the food industry, by means ofa particular freezing drum as disclosed in U.S. Pat. No. 5,036,673.

The process may further comprise the addition of an aqueous solutioncomprising one or more adjuvants and, optionally, a stabilizer to theliquid bulk antigen composition.

Alternatively, the process according to the present invention furthercomprises diluting a separate liquid bulk solution or emulsion of one ormore adjuvants by an aqueous solution comprising a stabilizer,subjecting the diluted adjuvant solution or emulsion to a process inorder to form regular droplets having a diameter of approximately fromabout 200 μm to about 1500 μm, subjecting the regular droplets tofreezing in a freezing medium to form frozen regular sphericalmicropellets or particles, drying the frozen regular sphericalmicropellets or particles to form dry regular spherical micropellets orparticles having a diameter from about 200 μm to about 1500 μm andblending and filling into a vial or other appropriate container togetherwith the antigen containing dry regular spherical micropellets.

The liquid bulk composition may further comprise one or more additionalantigens or antigenic preparations, each from a different pathogen orserotype of a pathogen, in order to obtain dry regular sphericalmicropellets or particles, each comprising two or more antigens orantigenic preparations from two or more different pathogens or serotypesof a pathogen.

Alternatively, the liquid bulk composition comprises an antigen orantigenic preparation from one single pathogen or serotype of apathogen, in order to obtain dry regular spherical micropellets orparticles, each comprising antigens or antigenic preparations from thesame pathogen or serotype of a pathogen. In this case the process mayoptionally further comprise the dosing, blending and filling into a vialor other appropriate container two or more types of dry regularspherical micropellets or particles, characterized in that each type ofmicropellets comprises antigens or antigenic preparations from two ormore different pathogens or serotypes of a pathogen.

The process according to the present invention may be furthercharacterized in that the drying occurs by the method of lyophilization(i.e. sublimation of ice and dissorption of bound water). Suitabledrying methods are also atmospheric freeze-drying, fluidized bed drying,vacuum rotary drum drying, stirred freeze-drying, vibrated freeze-dryingand microwave freeze-drying.

Advantageously, the stabilizer comprises a monosaccharide, such asmannose, an oligosaccharide, such as sucrose, lactose, trehalose,maltose or a sugar alcohol, such as sorbitol, mannitol or inositol, or amixture of two or more different of these before mentioned stabilizers,such as mixtures of sucrose and trehalose.

Advantageously, the concentration of carbohydrates, sugar alcohols andstabilizing excipients ranges from 2% (w/v) to limit of solubility inthe formulated liquid product. In general, the concentration ofcarbohydrates, sugar alcohols and stabilizing excipients ranges between5% (w/v) and 40% (w/v), 5% (w/v) and 20% (w/v) or 20% (w/v) and 40%(w/v). Examples for the concentration of a 1:1 mixture of sucrose andtrehalose in the formulated liquid product comprising e.g. tetanus ordiphtheria toxoids and aluminum gel (AlOOH) are 18.1% (w/v) and 17.5%(w/v), respectively.

The present invention particularly relates to a process for stabilizingan influenza vaccine composition comprising the steps of diluting aliquid bulk composition comprising an influenza antigen or an antigenicpreparation from a seasonal, pre-pandemic or pandemic influenza virusstrain by an aqueous solution comprising a carbohydrate and/or a sugaralcohol or a mixture of two or more different carbohydrates and/or sugaralcohols in order to obtain a 2% (w/v) to limit of solubility ofcarbohydrate and/or sugar alcohol content of the resulting dilutedcomposition, subjecting the diluted composition to a process in order toform regular droplets having a diameter of approximately from about 200μm to about 1500 μm, subjecting the regular droplets to freezing in afreezing medium to form frozen regular spherical micropellets orparticles and drying the frozen regular spherical micropellets orparticles to form dry regular spherical micropellets or particles havinga diameter from about 200 μm to about 1500 μm.

The liquid bulk composition comprising an influenza antigen or anantigenic preparation from a seasonal, pre-pandemic or pandemicinfluenza virus strain can be obtained by the method as disclosed inU.S. Pat. No. 6,048,537 (WO 96/05294).

The drying occurs advantageously by the method of lyophilization (i.e.sublimation of ice and dissorption of bound water). Further suitabledrying methods for the frozen micropellets are atmosphericfreeze-drying, fluidized bed drying, vacuum rotary drum drying, stirredfreeze-drying, vibrated freeze-drying, and microwave freeze-drying.

Advantageously, the carbohydrate is a disaccharide, particularlysucrose. Also suitable disaccharides are for example lactose, maltose ortrehalose. Also suitable for stabilizing the influenza vaccinecomposition are monosaccharides, such as mannose, or sugar alcohols,such as sorbitol, inositol or mannitol.

Advantageously, the concentration of the carbohydrates or sugar alcoholsranges between 5% and 40% (w/v), alternatively between 5% and 20% (w/v)or between 20% and 40% in the formulated liquid product. Examples forthe concentrations of e.g. sucrose in the formulated liquid product are2% (w/v), 10% (w/v) or 15.4% (w/v).

The liquid bulk composition may further comprise one or more additionalinfluenza antigens or antigenic preparations, each from a differentseasonal, pre-pandemic or pandemic influenza virus strain, in order toobtain dry regular spherical micropellets or particles, each comprisingtwo or more influenza antigens or antigenic preparations from two ormore different seasonal, pre-pandemic or pandemic influenza virusstrains. In this case the process optionally further comprises dosingand filling the dry regular spherical micropellets or particles into avial or other appropriate container.

Alternatively, the liquid bulk composition comprises one or moreinfluenza antigens or antigenic preparations from one seasonal,pre-pandemic or pandemic influenza virus strain, in order to obtain dryregular spherical micropellets or particles, each comprising influenzaantigens or antigenic preparations from the same seasonal, pre-pandemicor pandemic influenza virus strain. In this case the process mayoptionally further comprise dosing, blending and filling into a vial orother appropriate container two or more types of dry regular sphericalmicropellets or particles, characterized in that each type ofmicropellets comprises influenza antigens or antigenic preparations froma different seasonal, pre-pandemic or pandemic influenza virus strainthan the other type.

The process optionally further comprises the addition of an aqueoussolution comprising one or more adjuvants and, optionally, a stabilizerto the liquid bulk antigen composition.

Alternatively, the process according to the present invention furthercomprises diluting a separate liquid bulk solution or emulsion of one ormore adjuvants by an aqueous solution comprising a stabilizer,subjecting the diluted adjuvant solution or emulsion to a process inorder to form regular droplets having a diameter of approximately fromabout 200 μm to about 1500 μm, subjecting the regular droplets tofreezing in a freezing medium to form frozen regular sphericalmicropellets or particles, drying the frozen regular sphericalmicropellets or particles to form dry regular spherical micropellets orparticles having a diameter from about 200 μm to about 1500 μm andblending and filling into a vial or other appropriate container togetherwith the antigen containing dry regular spherical micropellets.

Particularly suitable adjuvants are for example liposomes, lipid ordetergent micelles or other lipidic particles, polymer nanoparticles ormicroparticles, soluble polymers, protein particles, mineral gels,mineral micro- or nanoparticles, polymer/aluminum nanohybrids, oil inwater or water in oil emulsions, saponin extracts, bacterial cell wallextracts, stimulators of innate immunity receptors, in particularnatural or synthetic TLR agonists, natural or synthetic NOD agonists andnatural or synthetic RIG agonists. A suitable adjuvant for the processaccording to the present invention for example is that disclosed in WO2007/006939.

The influenza virus strains are for example H5N1, H9N2, H7N7, H2N2, H7N1and H1N1 (Emergence of multiple genotypes of H5N1 avian influenzaviruses in Hong Kong SAR: Y Guan et al., 8950-8955, PNAS—Jun. 25,2002—vol 99 n° 13; H9N2 Influenza A Viruses from Poultry in Asia haveHuman Virus-like Receptor Specificity: M N Matrosovich, S Krauss and RWebster, Virology 281, 156-162 (2001); Evolution and Ecology ofInfluenza A Viruses: R Webster et al., Microbiological ReviewsMarch1992, p. 152-179). Alternatively, it could be an influenza strainselected from the group of the seasonal influenza virus strains.

The influenza antigen can be in a form selected from the groupconsisting of purified whole influenza virus, inactivated influenzavirus or sub-unit components of influenza virus, or a split influenzavirus.

The influenza antigen may be derived from cell culture. Alternatively,the influenza antigen is produced in embryonic eggs.

The present invention also concerns a stabilized dry vaccinecomposition, particularly a stabilized dry influenza vaccine compositionor stabilized dry vaccine compositions containing other antigens, suchas inactivated whole viruses or antigenic components of viruses,Influenza, Rotavirus, cytomegalo virus and Hepatitis A and B, and wholebacteria or bacterial protein or polysaccharide antigens, conjugated ornon-conjugated, such as Haemophilus influenzae, meningococcalpolysaccharides, tetanus, diphtheria, cellular and acellular pertussis,Botulism, Anthrax, C. Difficile in the form of dry regular sphericalmicropellets or particles having a diameter from about 200 μm to about1500 μm obtainable by the process according to the present invention.

Advantageously, each regular bead or particle comprises only one type ofantigen, for example one or more influenza antigens from only oneinfluenza virus strain or for example only Tetanus or only Diphtheriaantigens. Alternatively, each regular bead or particle comprises one ormore types of antigens, for example influenza antigens from one or moredifferent influenza virus strains or for example Tetanus and Diphtheriaantigens.

The composition may further comprise an adjuvant which is optionallycontained in separate dry regular spherical micropellets or particles.

The present invention further relates to a process for the preparation avaccine comprising the step of reconstitution of the stabilized dryvaccine composition, for example the before mentioned stabilized dryinfluenza vaccine composition in the form of dry regular sphericalmicropellets or particles in an aqueous solution. The aqueous solutionmay optionally comprise an adjuvant.

The present invention further relates to a vaccine kit, comprising afirst container containing a stabilized dry vaccine composition, forexample a stabilized dry influenza vaccine composition, in the form ofdry regular spherical micropellets or particles and a second containercontaining an aqueous solution for the reconstitution of the vaccine.The kit may further comprise a container containing dry regularspherical micropellets or particles comprising an adjuvant.Alternatively, the aqueous solution comprises an adjuvant.

The present invention also relates to a method of stockpiling a stabledry bulk of antigens wherein the antigen or the antigens is/arestabilized by the method described before and the resulting stabilizeddry vaccine composition (e.g. an influenza, diphtheria or tetanusvaccine composition) is reconstituted with an adequate solvent andoptionally formulated prior to liquid filling into vials or syringesafter storage in the form of dry regular spherical micropellets orparticles having a diameter from about 200 μm to about 1500 μm.

The process for thermo-stabilization of dry vaccine in micro-pellet formaccording to the present invention is explained in more detail in thefollowing.

In order to be processed by the micro-pellet technology, biologicalsubstances such as antigens require to be formulated in order to protectit against the physical and chemical stresses undergone throughout theprocess.

Formulated liquid products to be dried are obtained by mixing theconcentrated vaccine bulk (containing the antigen) and a stabilizingformulation comprising at least one carbohydrate and/or sugar alcohol,so that the formulated liquid product obtained contains the targetedamounts per ml of stabilizing excipients and antigens. The concentrationof carbohydrates, sugar alcohols and stabilizing excipients rangesbetween 2% (w/v) to limit of solubility in the formulated liquidproduct. To give an example, the concentration at the limit ofsolubility of sucrose in water at 20° C. is at about 66.7% (w/v).

FIG. 1 shows a flow chart of the general formulation and dryingprocedure.

The process as shown in FIG. 2 is used to generate the dry micropellets.

Prilling, also known as laminar jet break-up technique, is a well knowntechnique to generate calibrated droplets of liquid commonly used in thefield of biocatalysts and living cells immobilization (Hulst et al.,1985. A new technique for the production of immobilized biocatalyst andlarge quantities. Biotechnol. Bioeng. 27, 870-876; Gotoh et al., 1993.Mass production of biocatalyst-entrapping alginate gel particles by aforced oscillation method. Chem. Eng. Commun. 120, 73-84; Seifert andPhilips, 1997. Production of small, monodispersed alginate beads forcell immobilization. Biotechnol. Prog. 13, 562-568). Lord Rayleigh wasthe first to analyze instability of capillary jets coming out of anozzle and to propose a model to describe it (Rayleigh L, 1978. On thestability of jets. Proc. London Math. Soc. 10, 4-13) for Newtonianfluids. Weber (Weber C, 1931. Zum Zerfall eines Flüssigkeitsstrahles. Z.Angew. Math. Mech. 11, 136-154) extended the analysis including theeffect of the viscosity. The optimal wavelength for the fastest growingdisturbance and jet beak-up is given by:

$\lambda_{opt} = {\pi \cdot \sqrt{2} \cdot d_{j} \cdot \sqrt{1 + \frac{3\eta}{\sqrt{{\rho\sigma}\; d_{j}}}}}$where λ_(opt) is the optimal wave-length for jet break-up, d_(j) is thediameter of the jet, η is the viscosity of the fluid, ρ is the densityof the fluid and σ is the surface tension of the fluid. The diameter dof the droplets formed can be calculated by:d= ³√{square root over (1.5·d _(j) ²·λ_(opt))}

The frequency f to apply to the fluid to achieve the desired results isrelated to the jet velocity (and therefore the flow rate of the fluid)u_(j) and the wavelength by:

$\lambda = \frac{u_{j}}{f}$

Therefore; optimal conditions can be calculated knowing processparameters and fluid characteristics. However, a range of frequenciesand jet velocities exist to form uniform droplets depending on thenozzle diameter, rheology of the fluid and surface tension (Meesters G.,1992. Mechanisms of droplet formation. Delft University Press, Delft,NL). Suitable working frequencies can be also be determinedexperimentally by visual assessment of the stability of the dropletformation. Standard prilling equipments are equipped with lightstroboscope to observe the droplet formation: for a given product andgiven working conditions, one can adjust manually the frequency untilobserving a stable and still droplets chain with this stroboscope light.

Moreover, multinozzle systems have been developed for aseptic prillingapplications (Brandenberger H. et al. 1998. A new multinozzleencapsulation/immobilization system to produce uniform beads ofalginates. J. Biotechnol. 63, 73-80)

In conclusion, working frequencies can be determined both theoreticallyand experimentally depending on the available tools and knowledge of theproduct.

The prilling process is adapted to viscous liquids and saturated sugarsolutions. The maximum acceptable viscosity according to currentsuppliers is approximately 300 mPa·s. Therefore, formulation content canrange from very low concentration up to the limit of solubility of theselected excipients at room or processing temperature. Moreover, thetemperatures in the process tubing and in the nozzle have to becontrolled in order to avoid sugar or solvent crystallization beforedroplet formation. The person skilled in the art of formulation willadjust different excipient concentrations in the product in order toavoid non-controlled crystallization and viscosities above the givenlimit, taking into account eventual interactions between excipients.

The formulated liquid product is prilled in order to generate calibrateddroplets the diameter of which ranges from 200 μm to 1500 μm, with avery narrow size distribution. These droplets fall in a cryogenicchamber in which temperature is maintained below −110° C. either bydirect injection/nebulization of liquid nitrogen or by flowing countercurrently a very cold (T°<−110° C.) gas such as nitrogen, CO₂ or air.The droplets freeze during their fall in order to form calibrated frozenparticle. The minimum falling height to freeze the droplets (i.e. icecrystals formation that solidifies the pellets) can be minimized bylimiting super-cooling. Super-cooling can be reduced by seeding icenucleation in the droplets either by contact with liquid nitrogen fog ordroplets (direct injection of liquid nitrogen in the chamber) or withmicroscopic ice crystals (direct nebulization of water or superheatedsteam in the cold chamber).

These frozen particles are then collected and transferred on pre-cooledtrays at −50° C. and loading on the pre-cooled shelves of thefreeze-drier (−50° C.) in order to always keep the frozen pellets belowthe glass transition Tg′ of their cryo-concentrated phase (Tg′ value canrange from −10° C. to −45° C.) and to avoid any melting or aggregationof the particles. Once the freeze-drier is loaded, vacuum is pulled inthe freeze-drying chamber to initiate conventional freeze-drying(sublimation of the ice) of the pellets as know by the state of the art.The following freeze-drying parameters are an example of what can beused for a formulation which Tg′ ranges from −30° C. to −45° C.:

-   -   Primary drying: shelf temperature equal to −35° C., pressure        equal to 50 μbars during 10 h.    -   Secondary drying: shelf temperature equal to 20° C., pressure        equal to 50 μbars during 3 h.

Freeze-drying cycle has to be designed in order to get residualmoistures preferentially lower than 3%. However, the moisture contentcan be optimized at higher value, on a case by case basis, if thestability of the dry antigen requires it.

Other drying technologies such as atmospheric freeze-drying, fluidizedbed drying, vacuum rotary drum drying, stirred freeze-drying, vibratedfreeze-drying, microwave freeze-drying can be used to dry the pellets.

Dry pellets are then collected in bulk to be analyzed and stored.Storage conditions are suitable for dry, friable and hygroscopicparticles. Dry samples are rehydrated prior to analysis using a diluentwhich may contain water for injection, buffers, stabilizing excipientsand adjuvants. Dissolution is instantaneous.

Bulk dry pellets are then filled into vials using dry powder fillingtechnologies that are currently on the market.

Also, in a perspective of stockpiling a stable dry bulk of antigen, thepellets can be reconstituted with adequate solvent and formulated (ifneeded) prior to liquid filling into vials or into syringes. Thanks tothe stability of such pellets, the storage of the stockpile can beperformed at +5° C. or higher temperatures, which is more convenientthan for a frozen liquid material (−20° C. or lower). Finally,stockpiling dry bulk material is much less space consuming thanstockpiling filled dry products in their final container.In the case of adjuvanted vaccine, the stability during storage at +5°C. or higher and lower (freezing) temperature is often a problem whenantigens and adjuvants are in the same liquid state. As an example, thetable below describes the impact of a freeze-thawing cycle at −20° C. onoxyhydroxyl aluminum gel measured by dynamic light scattering:

Particles size (μm) mean d10 d50 d90 Initial +5° C. 8.9 2.6 4.9 10.3 1freeze-thawing cycle −20° C. 49.6 15.3 43.7 92.4

The micropellet technology allows overcoming stability problems with 3different strategies to be selected on a case by case basis:

1. Drying of the antigens alone under micropellet form and dissolutionwith the adjuvant (aluminum gel, emulsion, etc.) in the diluent. Thisstrategy is suitable when the antigen stability is increased undermicropellet form and when the adjuvant alone is highly thermostable

-   -   No interaction during shelf life    -   Increased stability of each phase when stored independently    -   Standardization of adsorption behavior by extemporaneous        dissolution of the dry antigen with the liquid adjuvant

2. Drying of the antigens with the adjuvant to stabilize both within thesame pellet and to stabilize the interaction between them by trappingthem in a glassy matrix in which all molecular motions and chemicalreactions are greatly hindered. This solid state allows to maintainthroughout storage (even at higher temperature) potency of the antigen,physical and immunological properties of the adjuvant and nature andforce of the interaction between the two.

3. Drying of the antigens and the separate drying of the adjuvant,followed by blending of the two before the filling or by a sequentialfilling. In some cases, stability of the adjuvant alone can be a problem(chemical stability of emulsions, physical stability of aluminum gels,liposomes and others . . . ) at the liquid state for long term storageat +5° C. or higher temperatures. The micropellet technology allowsimproving stability of the adjuvanted vaccine by generating separatemicropellets of antigen and adjuvant. Stabilizing formulation can beoptimized independently for each antigens and the adjuvant. micropelletsof antigens and adjuvants can be then subsequently filled into thevials. The separated solid state allows to avoid throughout storage(even at higher temperature) interactions between antigen and adjuvant,to maintain potency of the antigen and physical and immunologicalproperties of the adjuvant. In such a configuration, the content of thevial is more stable than any other configurations with either one of theantigens or in the adjuvant in the liquid state or when antigens andadjuvant are dried within the same pellets. Interactions betweenantigens and adjuvants are this way standardized as they occur onlyafter rehydration of the dry combination with a selected diluent whichmay comprise water for injection, buffers and stabilizing excipients.Interactions therefore exist only during the short period of timebetween rehydration and injection of the vaccine (fast interactions andvery short aging time). It is therefore possible to improve the overallstability of the two products (optimization of the formulation for eachproduct and not a compromise for the two together) and the vaccineitself, adjust the titer of one out of the two after the storage, tofacilitate the manufacturing process by separation of the two productsdrying.

In order to be processed by the micropellet technology, biologicalsubstances such as antigens and adjuvants require to be formulated inorder to protect them against the physical and chemical stressesundergone throughout the process.

In case of adjuvants, the stabilizing formulation has to maintain theirquality during processing (formulation, pelletizing, drying, storage,filling and rehydration)

If the strategy is to dry both antigens and adjuvants in the samemicro-pellet, formulated liquid product to be dried is obtained bymixing the concentrated vaccine bulk (containing the antigens), theconcentrated adjuvant bulk and a stabilizing formulation comprising atleast one carbohydrate and or sugar alcohol, so that the formulatedliquid product obtained contains the targeted amounts per ml ofstabilizing excipients, adjuvants and antigens. The concentration of thestabilizing excipients ranges between 2% (w/v) and limit of solubilityin the formulated liquid product.

FIG. 3 is a flow chart showing the formulation and drying procedurecomprising an adjuvant.

If the strategy is to dry antigens and adjuvants in separatemicropellets, the formulated liquid product to be dried (comprising theantigens) is obtained by mixing the concentrate vaccine bulk (comprisingthe antigens) and a stabilizing formulation, so that the formulatedliquid product obtained contains the targeted amounts per ml ofstabilizing excipients and antigens. The concentration of thecarbohydrates and the stabilizing excipients ranges between 2% (w/v) andlimit of solubility in the formulated liquid product.

The same way for the adjuvant, the formulated liquid product to be driedis obtained by mixing the concentrated adjuvant bulk and a stabilizingformulation, so that the formulated liquid product obtained contains thetargeted amounts per ml of stabilizing excipients and adjuvants. Thecarbohydrates and stabilizing excipients concentration ranges arebetween 2% (w/v) and limit of solubility in the formulated liquidproduct.

FIG. 4 is a flow chart showing the formulation and drying procedure inorder to produce the antigen containing micropellets, FIG. 5 is a flowchart showing the formulation and drying procedure in order to producethe adjuvant containing micropellets. FIG. 6 shows the combination ofthe different micropellets in order to complete the dry vaccine.

The general process for the generation of the dry micropellets is shownin FIG. 2. It is applicable independently from the chosen strategy forthe adjuvanted vaccine.

Fast freezing kinetics of the micro-pellet technology is also adaptedfor drying adjuvants. As an example, the graph in FIG. 9 shows sizedistribution results after drying of an aluminum phosphate gel inpresence of a carbohydrate and using the micro-pellet technology. It canbe seen that drying and rehydration has no significant negative effecton the size distribution of the adjuvant particles.

Suitable adjuvants that may be considered are exemplified in thefollowing:

1) The particulate adjuvants such as: liposomes and in particularcationic liposomes (e.g. DC-Chol, see e.g. US 2006/0165717, DOTAP, DDABand 1,2-Dialkanoyl-sn-glycero-3-ethylphosphocholin (EthylPC) liposomes,see U.S. Pat. No. 7,344,720), lipid or detergent micelles or other lipidparticles (e.g. Iscomatrix from CSL or from Isconova, virosomes andproteocochleates), polymer nanoparticles or microparticles (e.g. PLGAand PLA nano- or microparticles, PCPP particles, Alginate/chitosanparticles) or soluble polymers (e.g. PCPP, chitosan), protein particlessuch as the Neisseria meningitidis proteosomes, mineral gels (standardaluminum adjuvants: AlOOH, AlPO₄), microparticles or nanoparticles (e.g.Ca₃(PO₄)₂), polymer/aluminum nanohybrids (e.g. PMAA-PEG/AlOOH andPMAA-PEG/AlPO₄ nanoparticles) O/W emulsions (e.g. MF59 from Novartis,AS03 from GlaxoSmithKline Biologicals) and W/O emulsion (e.g. ISA51 andISA720 from Seppic, or as disclosed in WO 2008/009309). For example, asuitable adjuvant emulsion for the process according to the presentinvention is that disclosed in WO 2007/006939.

2) The natural extracts such as: the saponin extract QS21 and itssemi-synthetic derivatives such as those developed by Avantogen,bacterial cell wall extracts (e.g. micobacterium cell wall skeletondeveloped by Corixa/GSK and micobaterium cord factor and its syntheticderivative, trehalose dimycholate).

3) The stimulators of innate immunity receptors such as: natural orsynthetic TLR agonists (e.g. synthetic lipopeptides that stimulateTLR2/1 or TLR2/6 heterodimers, double stranded RNA that stimulates TLR3,LPS and its derivative MPL that stimulate TLR4, E6020 and RC-529 thatstimulate TLR4, flagellin that stimulates TLR5, single stranded RNA and3M's synthetic imidazoquinolines that stimulate TLR7 and/or TLR8, CpGDNA that stimulates TLR9, natural or synthetic NOD agonists (e.g.Muramyl dipeptides), natural or synthetic RIG agonists (e.g. viralnucleic acids and in particular 3′ phosphate RNA).

These adjuvants may also be used in combination. Preferred combinationsare those between the particulate adjuvants and natural extracts andthose between particulate adjuvants and stimulators of innate immunityreceptors.

For the following examples, the prilling apparatus IE-50R EncapsulatorResearch, from Inotech (CH) and a 300 μm nozzle head were used togenerate the micropellets.

EXAMPLE 1 Manufacturing of Thermo-Stable Dry Vaccine Under MicropelletForm Containing Flu Antigens

This study compared the thermo-stability of the current liquid trivalentflu vaccine to dry formulations of this same vaccine processed with themicro pellet technology. Trivalent flu vaccine contains 30 μg/ml of eachA/Salomon, B/Malaysia and A/Wisconsin strains in the vaccinal buffer.Formulated liquid products to be dried were obtained by mixing onevolume of trivalent flu vaccine with one volume of a stabilizingformulation comprising at least one carbohydrate which concentrationranges between 4% (w/v) and limit of solubility. This corresponds to aconcentration range from 2% to 32% (weight by volume) in the formulatedliquid product to be dried. Two formulations were evaluated: SG1 and SG2which compositions are given in tables 1 and 2:

TABLE 1 SG1 COMPONENTS quantity for 1000 ml Sucrose 200 g Adjustment pH@ 7.0 +/− 0.2 (NaOH, HCl) Water PPI 1000 ml

TABLE 2 SG2 COMPONENTS quantity for 1000 ml Sucrose 200 g Glutamine 2 gUrea 10 g Dextrane 70 10 g Adjustment pH @ 7.0 +/− 0.2 (NaOH, HCl) WaterPPI 1000 ml

FIG. 7 shows a flowchart of the formulation and drying procedure.

FIG. 2 shows the process that was used to generate the dry micropelletsof formulations SG1 and SG2.

Formulated liquid product SG1 and SG2 were prilled in order to generatecalibrated droplets. Prilling parameters for this formulation and a 300μm nozzle head were:

-   -   Product flow rate: 8 ml/min    -   nozzle frequency: 954 Hz

These droplets fell in a cryogenic chamber in which the temperature wasmaintained below −110° C. by direct injection of liquid nitrogen or byflowing countercurrent very cold gas (t°<−110° C.). The droplets frozeduring their fall and formed calibrated frozen particles.

These frozen particles were then transferred on pre-cooled trays at −50°C. and loaded on the pre-cooled shelves of the freeze-drier (−50° C.) inorder to always keep the frozen pellets below their glass transition(which was evaluated between −30° C. and −40° C.) and to avoid anymelting or aggregation of the particles. Once the freeze-dryer wasloaded, vacuum was pulled in the freeze-drying chamber to initiateconventional freeze-drying of the pellets as know by the state of theart. For these formulations, the following freeze-drying parameters wereused: Primary drying: shelf temperature equal to −35° C., pressure equalto 50 μbars during 10 h. Secondary drying: shelf temperature equal to20° C., pressure equal to 50 μbars during 3 h. Residual moisture of themicropellets was below 2% for both SG1 and SG2 formulations.

FIG. 8 gives an idea of the very narrow size distribution obtained withthis pelletizing and drying technology.

Therefore, 3 products were available for a thermo-stability study: Themarketed trivalent flu vaccine (VAXIGRIP®, Sanofi Pasteur), the drymicropellets SG1 formulation (trivalent flu vaccine) and the drymicropellets SG2 formulation (trivalent flu vaccine).

Samples of each of these 3 formulations were exposed to different timeat 37° C. and 45° C. Potency (μg of antigen/ml) was then measured foreach sample by SRD (SRID) method (M. S. Williams, VeterinaryMicrobiology, 37 (1993) 253-262). Dry samples were re-hydrated usingwater for injection prior to analysis. Dissolution was instantaneous.The tables 3, 4 and 5 summarize the obtained results. Results areexpressed in mean value μg/ml of antigen.

TABLE 3 A/Salomon Stability at Stability 37° C. (days) at 45° C. (days)0 7 30 90 0 7 Liquid formulation 29.8 24.3 12.1 29.8 16.4 micropelletsSG1 23.2 22.5 24.9 27 23.2 24.3 micropellets SG2 26.5 24.7 28.2 29.426.5 25.8

TABLE 4 B/Malaysia Stability at Stability 37° C. (days) at 45° C. (days)0 7 30 90 0 7 Liquid formulation 30 21.4 14.4 30 16.3 micropellets SG125.4 25.7 24.9 28.2 25.4 24 micropellets SG2 26.6 25.4 27.2 30.4 26.626.7

TABLE 5 A/Wisconsin Stability at Stability 37° C. (days) at 45° C.(days) 0 7 30 90 0 7 Liquid formulation 30 24.7 13.2 30 16 micropelletsSG1 25.4 24.6 25.5 26.8 25.4 25 micropellets SG2 28.7 26 28.4 30.7 28.727.8

These results show that the dried formulations SG1 and SG2 in themicro-pellet form are much more stable than the current liquidformulation. No significant antigenicity loss was measured after up to 3months at 37° C. and 1 week at 45° C.

EXAMPLE 2 Manufacturing of Thermo-Stable Dry Vaccine Under MicropelletForm Containing Flu H5N1 (Indonesia) Antigens Adjuvanted with AluminumOxyhydroxide Gel

This study compared the thermo-stability of the liquid H5N1 Indonesiaflu vaccine to dry formulations of this same vaccine processed with themicropellet technology according to the present invention.

The vaccine contains 65.4 μg/ml of H5N1 Indonesia strain in the vaccinalbuffer, adjuvanted by aluminum oxyhydroxide gel.

Formulated liquid product to be dried was obtained by mixing one volumeof H5N1 vaccine with one volume of the stabilizing formulation SG1. (cf.Example 1 for SG1 composition)

FIG. 10 shows a flowchart of the formulation and drying procedure.

Formulated liquid product SG1 was prilled in order to generatecalibrated droplets. Prilling parameters for this formulation and a 300μm nozzle head were:

-   -   Product flow rate: 8 ml/min    -   nozzle frequency: 916 Hz

These droplets fell in a cryogenic chamber in which temperature wasmaintained below −110° C. by direct injection of liquid nitrogen or byflowing countercurrent very cold gas (t°<−110° C.). The droplets frozeduring their fall and formed calibrated frozen particles.

These frozen particles were then transferred on pre-cooled trays at −50°C. and loading on the pre-cooled shelves of the freeze-drier (−50° C.)in order to always keep the frozen pellets below their glass transition(which was evaluated between −30° C. and −40° C.) and to avoid anymelting or aggregation of the particles. Once the freeze-drier wasloaded, vacuum was pulled in the freeze-drying chamber to initiateconventional freeze-drying of the pellets as know by the state of theart. For these formulations, the following freeze-drying parameters wereused: Primary drying: shelf temperature equal to −35° C., pressure equalto 50 μbars during 10 h. Secondary drying: shelf temperature equal to20° C., pressure equal to 50 μbars during 3 h. Residual moisture of themicropellets was below 2%.

Micropellets samples were exposed 7 days at 37° C. and 45° C. Potency(μg of antigen/ml) was then measured for each sample by SRD method. Drysamples were rehydrated using water for injection prior to analysis.Dissolution was instantaneous. The table 6 summarizes the obtainedresults. Results are expressed in mean value μg/ml of antigen; N.D.stands for “Non Detectable Antigenicity”.

TABLE 6 SRD Titers μg/ml Liquid formulation Micropellet SG1 To 65.4 53.67 d @ 37° C. 52.5 51.4 7 d @ 45° C. <7.0 47.9 7 d @ 55° C. N.D. 44.9

These results show that the dried formulation SG1 in the micro-pelletform is much more stable than the current liquid formulation. Nosignificant antigenicity loss was measured after 1 week at 37° C. and at45° C. A loss of approximately 15% was observed after 1 week at 55° C.,which is close to the precision of the assay itself.

The impact of this drying process and the rehydration of the pellets onthe adjuvant properties were evaluated by measuring the aluminumoxyhydroxide particle size distribution in the formulated bulk beforedrying, and after dissolution of the pellets using a particle sizeanalyzer Malvern MasterSizer 2000. The results are given FIG. 11. Weobserved that this process did not induced aggregation of the gel.

FIG. 12 also shows that the gel's size is maintained afterthermostability incubation for at least 7 days at 37° C., 45° C. and 55°C., and demonstrates the stability of alum gel adjuvant in a micropelletform.

This example confirms the applicability of this technology to alum geladjuvanted Flu antigens, and most generally, the feasibility to dryadjuvanted antigens with alum gels using the micropellet technology.

EXAMPLE 3 Manufacturing of Thermo-Stable Dry Vaccine Under MicropelletForm Containing Non-Adjuvanted Flu H5N1 (Indonesia) and Rehydration withan Adjuvant Emulsion

This study compared the thermo-stability of the liquid H5N1 Indonesiaflu vaccine to dry formulations of this same vaccine processed with themicropellet technology.

The vaccine contains 176 μg/ml of H5N1 Indonesia strain in the vaccinalbuffer.

Formulated liquid product to be dried was obtained by mixing the H5N1vaccine with the stabilizing formulation SG1, in order to target thedesired antigen concentration and stabilizer contents. The formulationSG1 was evaluated (cf. Example 1 for SG1 composition)

FIG. 13 shows a flowchart of the formulation and drying procedure.

Formulated liquid product was prilled in order to generate calibrateddroplets. Prilling parameters for this formulation and a 300 μm nozzlehead were:

-   -   Product flow rate: 8 ml/min    -   nozzle frequency: 997 Hz        These droplets fell in a cryogenic chamber in which the        temperature was maintained below −110° C. by direct injection of        liquid nitrogen or by flowing countercurrent very cold gas        (t°<−110° C.). The droplets froze during their fall and formed        calibrated frozen particles.

These frozen particles were then transferred on pre-cooled trays at −50°C. and loading on the pre-cooled shelves of the freeze-drier (−50° C.)in order to always keep the frozen pellets below their glass transition(which was evaluated between −30° C. and −40° C.) and to avoid anymelting or aggregation of the particles. Once the freeze-drier wasloaded, vacuum was pulled in the freeze-drying chamber to initiateconventional freeze-drying of the pellets as know by the state of theart. For these formulations, the following freeze-drying parameters wereused: Primary drying: shelf temperature equal to −35° C., pressure equalto 50 μbars during 10 h. Secondary drying: shelf temperature equal to20° C., pressure equal to 50 μbars during 3 h. Residual moisture of themicropellets was below 2%.

In parallel, the same formulated product was generated, filled intovials and freeze-dried normally in a standard freeze-dryer. Filled vialswere loaded on pre-cooled shelves at 5° C.; the product was then frozendown to −50° C. at 1° C./min and vacuum was pulled to initiatesublimation. Primary drying parameters were: shelf temperature equal to−18° C., pressure equal to 50 μbars during 16 h. Secondary dryingparameters were: shelf temperature equal to 37° C., pressure equal to 50μbars during 2 h.

Residual moisture of the freeze-dried product was also below 2%.

Micropellet and liquid samples were exposed to different time at 37° C.,45° C. and 55° C. Freeze-dried samples were exposed at 37° C. and 55° C.Potency (μg of antigen/ml) was then measured for each sample by SRDmethod. Dry samples were rehydrated using water for injection (WFI)prior to analysis. Dissolution was instantaneous. The tables 7, 8 and 9summarize the obtained results respectively for a standard liquidformulation, dried micropellets and the freeze-dried product. Resultsare expressed in mean value μg/ml of antigen. Initial SRD Titer at Tocorresponds to the measured titer after reconstitution of themicropellets after processing.

TABLE 7 Initial SRD Titer: To = 14.1 μg/ml Liquid H5N1 Stability studyTime SRD Titers μg/ml 7 days 1 month 3 months Thermostability at 37° C.11.6 5 <5 Thermostability at 45° C. 3.4 <5 <5 Thermostability at 55° C.<5 <5 <5

TABLE 8 Dry micropellets H5N1 Initial SRD Titer: To = 47.2 μg/mlStability study Time SRD Titers μg/ml, Rehydration WFI 7 days 1 month 3months Thermostability at 37° C. 53 47.3 50.1 Thermostability at 45° C.51.6 47 41.1 Thermostability at 55° C. 49.5 45.4 47.3

TABLE 9 Initial SRD Titer: To = 39.4 μg/ml Freeze-dried H5N1 Stabilitystudy Time SRD Titers μg/ml, Rehydration WFI 14 days 1 monthThermostability at 37° C. 36.9 38.1 Thermostability at 55° C. 35.6 35.1

These results show that the dried formulation SG1 in the micro-pelletform is much more stable than the current liquid formulation. Nosignificant antigenicity loss was measured after 3 months at 37° C., 45°C. and 55° C. Results given table 9 confirm that standard freeze-dryingalso provides good thermostability.

Moreover, the data given table 10 show that no antigenicity loss wasmeasured after 9 months at +5° C. These results are very promising forlong term stability at +5° C. and room temperature over a several yeartime period.

The feasibility of rehydrating the H5N1 micropellets with an emulsionhas been studied. The emulsion used for this study is the one describedin the patent application WO 2007/006939. The same experimental plan astable 8 was performed but samples were rehydrated with the emulsionrather than water for injection. The results are given table 10 andtable 11:

TABLE 10 H5N1 micropellet H5N1 micropellet formulation formulation To +9months at (μg/ml)at To (μg/ml) + 5° C. After Drying. 47.2 54.8Rehydration WFI After Drying. 49.2 51 Rehydration Emulsion

TABLE 11 Dry H5N1 Stability study Initial SRD Titer: To = 49.2 μg/ml SRDTiters μg/ml. Time Rehydration Emulsion 7 days 1 month 3 monthsThermostability at 37° C. 46.5 42.6 47.8 Thermostability at 45° C. 43.146.1 46.5 Thermostability at 55° C. 41.8 42.7 41.7

Table 10 proves that the dissolution of the micropellets with anemulsion as adjuvant does not impact the stability of the antigen andtherefore its recovery. Table 11 confirms that this statement is alsoverified after thermostability incubation of the micropellets as allmeasured titers are comparable with titers measured after dissolutionwith water for injection.

FIG. 14 shows a comparison of the size of the emulsion beforerehydration of these micropellets and after dissolution. Thesuperposition of the two size distributions confirms that the size ofthe emulsion, and therefore its integrity, is not significantly alteredby the rehydration of a freeze-dried matrix. The size distributionremains monomodal and centered on 100 nm. Moreover, FIG. 15 confirms thestability of the size of the emulsion after dissolution of themicropellets and over a one hour storage period at room temperature.

This study confirms that, thanks to the micropellet technology, it ispossible to use the adjuvant as a dissolution buffer and to avoid allinteraction between the adjuvant and the antigen during the shelf lifeof the product.

EXAMPLE 4a Manufacturing of a Thermo-Stable Dry Vaccine UnderMicropellet Form Containing Non-Adjuvanted Flu H5N1 (Indonesia) withDifferent Sugars Used as Stabilizers

This study shows the preparation of 3 stabilized dry influenza vaccinecompositions each comprising a different disaccharide (trehalose,lactose and maltose respectively) and the thermo-stability of such drycompositions processed with the micropellet technology.

The vaccine contains 131 μg/ml of H5N1 Indonesia strain in the vaccinalbuffer.

The formulated liquid products to be dried were obtained by mixing theH5N1 vaccine with the stabilizing formulations SG5, SG6 and SG7,respectively (see tables 12a, 12b and 12c), in order to target thedesired antigen concentrations and stabilizer contents.

TABLE 12a SG5 COMPONENTS quantity for 1000 ml Trehalose 200 g AdjustmentpH @ 7.0 +/− 0.2 (NaOH, HCl) Water PPI 1000 ml

TABLE 12b SG6 COMPONENTS quantity for 1000 ml Lactose 200 g AdjustmentpH @ 7.0 +/− 0.2 (NaOH, HCl) Water PPI 1000 ml

TABLE 12c SG7 COMPONENTS quantity for 1000 ml Maltose 200 g AdjustmentpH @ 7.0 +/− 0.2 (NaOH, HCl) Water PPI 1000 ml

FIG. 13 shows a flowchart of the principle of the formulation and dryingprocedure with SG1 stabilizer as an example. Identical flowchart wasused with SG5, SG6 and SG7 to obtain a disaccharide concentration of15.4% (w/v) in the formulated product prior to prilling.

Each of the 3 formulated liquid products was prilled in order togenerate calibrated droplets. Prilling parameters for these formulationand a 300 μm nozzle head were:

-   -   Product flow rate: 8 ml/min    -   nozzle frequency: ranging from 994 Hz and 1001 Hz depending on        the formulation

These droplets fell in a cryogenic chamber in which the temperature wasmaintained below −110° C. by direct injection of liquid nitrogen or byflowing countercurrent very cold gas (t°<−110° C.). The droplets frozeduring their fall and formed calibrated frozen particles.

These frozen particles were then transferred on pre-cooled trays at −50°C. and loading on the pre-cooled shelves of the freeze-drier (−50° C.)in order to always keep the frozen pellets below their glass transition(which was evaluated between −30° C. and −40° C.) and to avoid anymelting or aggregation of the particles. Once the freeze-drier wasloaded, vacuum was pulled in the freeze-drying chamber to initiateconventional freeze-drying of the pellets as know by the state of theart. For these formulations, the following freeze-drying parameters wereused: Primary drying: shelf temperature equal to −35° C., pressure equalto 50 μbars during 10 h. Secondary drying: shelf temperature equal to20° C., pressure equal to 50 μbars during 3 h. Residual moisture of themicropellets was below 2%.

Micropellets samples were exposed to different time at 37° C. and 55° C.Potency (μg of antigen/ml) was then measured for each sample by SRDmethod. Dry samples were rehydrated using water for injection (WFI)prior to analysis. Dissolution was instantaneous. The tables 12d, 12eand 12f summarize the obtained thermostability results respectively foreach of the 3 compositions in the form of dried micropellets. Resultsare expressed in mean value μg/ml of antigen. Initial SRD Titer at Tocorresponds to the measured titer after reconstitution of themicropellets after processing.

TABLE 12d Dry micropellets Initial SRD Titer: To = 61.5 μg/ml H5N1 + SG5Stability study Time SRD Titers μg/ml, Rehydration WFI 14 days 1 monthThermostability at 37° C. 57.7 58.7 Thermostability at 55° C. 57.2 58.9

TABLE 12e Dry micropellets Initial SRD Titer: To = 60.2 μg/ml H5N1 + SG6Stability study Time SRD Titers μg/ml, Rehydration WFI 14 days 1 monthThermostability at 37° C. 53.0 50.5 Thermostability at 55° C. 52.4 53.1

TABLE 12f Dry micropellets Initial SRD Titer: To = 52.9 μg/ml H5N1 + SG7Stability study Time SRD Titers μg/ml, Rehydration WFI 14 days 1 monthThermostability at 37° C. 52.8 49.6 Thermostability at 55° C. 50.4 51.2

These results confirm a similar stability profile for a wide range ofsaccharide excipients used as stabilizer.

EXAMPLE 4b Manufacturing of a Thermo-Stable Dry Vaccine UnderMicropellet Form Containing Non-Adjuvanted Flu H5N1 (Indonesia) with 2%(W/V) Sucrose in the Formulated Bulk Ready to be Processed

The vaccine contains 176 μg/ml of H5N1 Indonesia strain in the vaccinalbuffer.

The formulated liquid product to be dried was obtained by mixing theH5N1 vaccine with the stabilizing formulation SG8, in order to targetthe desired antigen concentration and a stabilizer content of 2% (w/v).Table 12g shows the composition of the stabilizing formulation SG8.

TABLE 12g SG8 COMPONENTS quantity for 1000 ml Sucrose 26 g Adjustment pH@ 7.0 +/− 0.2 (NaOH, HCl) Water PPI 1000 ml

The micropellets made from the formulated liquid bulk containing 2% w/vsucrose (liquid bulk formulated with SG8) were obtained as described inexample 4a. FIG. 16 shows a flowchart of the formulation and dryingprocedure.

The micropellet samples were exposed to different time at 37° C. and 55°C. Potency (μg of antigen/ml) was then measured for each sample by SRDmethod. Dry samples were rehydrated using water for injection (WFI)prior to analysis. Dissolution was instantaneous. Table 12h shows theobtained results for the dried micropellets. The results are expressedin mean value μg/ml of antigen. Initial SRD Titer at To corresponds tothe measured titer after reconstitution of the micropellets right afterprocessing.

TABLE 12h Dry micropellets Initial SRD Titer: To = 39.6 μg/ml H5N1 + SG8Stability study Time SRD Titers μg/ml, Rehydration WFI 14 days 1 monthThermostability at 37° C. 32.9 33.6 Thermostability at 55° C. 34.71 34.0

EXAMPLE 5 Study of the Impact of Micropellet Processing on AdjuvantedDiphtheria Toxoid (Dt) and Tetanus Toxoid (Tt) Vaccines

The first part of this study evaluated the stability of TetanusTt and Dtantigens freeze-dried in a micropellet form either with or withoutpre-adsorption on aluminum gel ALOOH. Five formulations were prepared asdescribed below.

Formulated liquid products to be dried containing DiphtheriaDt orTetanusTt in presence of aluminum gel were obtained by mixing a givenvolume of Diphtheria or Tetanus toxoid concentrated bulk with aluminumgel and a stabilizer in order to obtain the following composition:

-   -   Dt (Diphteria toxoid) formulated product: 200 Lf/ml of antigen        and 0.8 mg/ml of aluminum gel    -   Tt (Tetanus toxoid) formulated product: 40 Lf/ml of antigen and        0.8 mg/ml of aluminum gel

FIG. 17 shows a flowchart of the formulation and drying procedure ofthese two formulations

Formulated liquid products to be dried containing DiphtheriaDt orTetanusTt without aluminum gel were obtained by mixing a given volume ofDiphtheria or Tetanus toxoid concentrated bulk with a stabilizer inorder to obtain the following composition:

-   -   Dt (Diphteria toxoid) formulated product: 500 Lf/ml of antigen    -   Tt (Tetanus toxoid) formulated product: 100 Lf/ml of antigen

FIG. 18 shows a flowchart of the formulation and drying procedure ofthese two formulations.

Antigen contents for Diphtheria toxoid and Tetanus toxoid were measuredusing the rocket immuno-electrophoresis test, as performed by the stateof the art.

Formulated liquid product to be dried containing only aluminum gel isobtained by mixing a given volume aluminum gel with a stabilizer inorder to obtain the following composition:

-   -   Aluminum gel ALOOH formulated product: 2.4 mg/ml

FIG. 19 shows a flowchart of the formulation and drying procedure ofthis formulation. The composition of the stabilizer used for thisexperiment, called SDT-1, is given in the table below:

TABLE 13 SDT-1 Quantity for 1000 ml Sucrose 100 g Trehalose 100 g Tris50 mM 6,055 g pH adjustment 7.0 +/− 0.2 (NaOH, HCl) Water for injection1000 mL

Formulated liquid products were prilled in order to generate calibrateddroplets. Prilling parameters for these formulations and a 300 μm nozzlehead are summarized in the table below:

TABLE 14 Diphtheria Tetanus Diphtheria Tetanus toxoid + toxoid + toxoidtoxoid Al gel Al gel Al gel Flow rate 8 ml/min 8 ml/min 8 ml/min 8ml/min 8 ml/min Frequency 1487 1543 1223 1282 1479 (Hz)These droplets fell in a cryogenic chamber in which temperature wasmaintained below −110° C. by direct injection of liquid nitrogen or byflowing countercurrent very cold gas (t°<−110° C.). The droplets frozeduring their fall and formed calibrated frozen particles.

These frozen particles were then transferred on pre-cooled trays at −50°C. and loading on the pre-cooled shelves of the freeze-drier (−50° C.)in order to always keep the frozen pellets below their glass transition(which was evaluated between −30° C. and −40° C.) and to avoid anymelting or aggregation of the particles. Once the freeze-drier wasloaded, vacuum was pulled in the freeze-drying chamber to initiateconventional freeze-drying of the pellets as know by the state of theart. For these formulations, the following freeze-drying parameters wereused: Primary drying: shelf temperature equal to −35° C., pressure equalto 50 μbars during 10 h. Secondary drying: shelf temperature equal to20° C., pressure equal to 50 μbars during 3 h. Residual moisture of themicropellets was below 2%.

Micropellets samples were exposed at 37° C. and 55° C. Potency (Lf/ml)was measured for each sample by rocket immuno-electrophoresis test. Drysamples were re-hydrated using water for injection prior to analysis.Dissolution was instantaneous.

Diphtheria toxoid stability results:

TABLE 15 Diphtheria Toxoid Time (days) Target = 100 Lf/ml 0 14 30 90Stability at 37° C. (Lf/ml) 98.5 100.3 100 86.7 Stability at 55° C.(Lf/ml) 98.5 102.2 104 87.6These results confirm no significant loss after up to 1 month at 37° C.and 55° C. and only about 13% loss after 3 months, which is at the limitof significance. Moreover, incubation of micropellets containingDiphtheria toxoid and alum gel showed that at all time points and alltested temperatures, 100% of the antigen remained adsorbed to the gelafter dissolution. The observed stability of the gel size distributionafter dissolution and thermo-stability study showed similar results asfor flu H5N1 adjuvanted with alum gel (see example 2).

Tetanus toxoid stability results:

TABLE 16 Tetanus Toxoid Time (days) Target = 20 Lf/ml 0 14 30 90Stability at 37° C. (Lf/ml) 18.1 21.8 20.1 17.1 Stability at 55° C.(Lf/ml) 18.1 21.8 20.9 18

These results confirm no significant loss after up to 3 months at 37° C.and 55° C. Moreover, incubation of micropellets containing Tetanustoxoid and alum gel showed that at all time points and all testedtemperatures, 100% of the antigen remained adsorbed to the gel afterdissolution. The observed stability of the gel size distribution afterdissolution and thermo-stability study showed similar results as for fluH5N1 adjuvanted with alum gel (see example 2).

These data confirm that the micropellet process allows obtainingthermostable dry adjuvanted or non-adjuvanted Diphtheria and Tetanustoxoid vaccines, when properly formulated, in terms of potency,adsorption state and adjuvant quality, up to 3 months at 55° C. withoutsignificant degradation. Moreover, aluminum gel could successfully befreeze-dried using the micropellet technology maintaining a comparablesize distribution and avoiding massive aggregation.

EXAMPLE 6 Study of the Impact of Micropellet Processing on Aluminum GelCharacteristics: Interactions with T (Tetanus Toxoid)

In this example, micropellets generated in example 5 were used. The goalof this study was to evaluate the impact of the micropellet processingon aluminum gel AlOOH adsorption capacity.

In 3 ml vials, a constant quantity of aluminum gel AlOOH of 0.3 mg,initially in a liquid or micropellet form, was mixed with differentquantity of Tetanus toxoid (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 et 0.75 mgtotal), initially in a liquid or micropellet form as well.

5 experiments were performed:

-   -   Series 1: liquid mix of liquid aluminum gel AlOOH and bulk        Tetanus toxoid antigen, in the absence of stabilizer    -   Series 2: liquid mix of dissolved Aluminum gel AlOOH        micropellets and bulk Tetanus toxoid antigen    -   Series 3: liquid mix of liquid aluminum gel AlOOH, bulk Tetanus        toxoid antigen and stabilizer    -   Series 4: liquid mix of liquid aluminum gel AlOOH and dissolved        Tetanus toxoid micropellets    -   Series 5: Liquid mix of dissolved aluminum gel AlOOH        micropellets and dissolved Tetanus toxoid micropellets        Water and stabilizer contents were adjusted before dissolution        of the micropellets in order to obtain a rehydrated solution        strictly identical in all stabilizer containing formulations        (Series 2 to 5).

After mixing the liquid products, the vials were incubated at roomtemperature in a wheel agitator during 2 hours and then centrifuged at3000 rpm during 5 minutes. The non adsorbed Tetanus toxoid wasquantified in the supernatant by Micro Bradford technique (Bio Radprotein assay). A Tetanus toxoid reference was tested for each series ofsamples in order to have a quantitative assay. The obtained results aresummarized in the table below:

TABLE 17 Presence Tetanus toxoid adsorption Average of stabilizercapacity mg toxoid/mg gel mg toxoid/mg gel Series 1 No 0.49 0.61 ± 0.120.72 Series 2 yes 0.67 0.60 ± 0.07 0.54 Series 3 yes 0.82 0.76 ± 0.220.54 0.92 Series 4 yes 0.57 0.64 ± 0.08 0.72 Series 5 yes 0.59 0.86 ±0.26 1.06 0.91

These results confirm that the presence of a stabilizer does not haveany significant negative impact on the adsorption capacity of the gel.Moreover, micropellet processing does not impact significantly, takinginto account the variability of the method, adsorption capacity of alumgel when applied on Tetanus toxoid and/or alum gel.

The invention claimed is:
 1. A process for producing a stabilizedinfluenza vaccine composition comprising: a) diluting a liquid bulkantigen composition comprising an influenza antigen or an antigenicpreparation from a seasonal, pre-pandemic or pandemic influenza virusstrain with an aqueous solution comprising a carbohydrate, a sugaralcohol, or a mixture thereof in order to obtain a diluted vaccinecomposition solution wherein the concentration of the carbohydrate,sugar alcohol, or mixture thereof is from 2% (w/v) to the limit ofsolubility, b) prilling the diluted vaccine composition solution to formregular droplets of the vaccine having a diameter of approximately fromabout 200 μm to about 1500 μm, c) subjecting the regular droplets of thevaccine to freezing to form frozen regular spherical micropellets orparticles of the vaccine, and d) drying the frozen regular sphericalmicropellets or particles of the vaccine to form dry regular sphericalmicropellets or particles of the vaccine having a diameter from about200 μm to about 1500 μm.
 2. The process as claimed in claim 1, whereinthe drying occurs by lyophilization.
 3. The process as claimed in claim1, wherein the drying occurs by atmospheric freeze-drying, fluidized beddrying, vacuum rotary drum drying, stirred freeze-drying, vibratedfreeze-drying, or microwave freeze-drying.
 4. The process as claimed inclaim 1, wherein the carbohydrate is a disaccharide.
 5. The process asclaimed in claim 4, wherein the carbohydrate is sucrose.
 6. The processas claimed in claim 1, wherein the liquid bulk antigen compositionfurther comprises one or more additional influenza antigens or antigenicpreparations, each antigen in the composition being from a differentseasonal, pre-pandemic or pandemic influenza virus strain, in order toobtain a vaccine in the form of dry regular spherical micropellets orparticles, each comprising two or more influenza antigens or antigenicpreparations from two or more different seasonal, pre-pandemic orpandemic influenza virus strains.
 7. The process as claimed in claim 6,further comprising dosing and filling the dry regular sphericalmicropellets or particles into a vial or other appropriate container. 8.The process as claimed in claim 1, wherein the liquid bulk antigencomposition comprises one or more influenza antigens or antigenicpreparations from one seasonal, pre-pandemic or pandemic influenza virusstrain, in order to obtain a vaccine in the form of dry regularspherical micropellets or particles, each comprising influenza antigensor antigenic preparations from the same seasonal, pre-pandemic orpandemic influenza virus strain.
 9. The process as claimed in claim 8,further comprising dosing, blending and filling into a vial or otherappropriate container two or more types of dry regular sphericalmicropellets or particles, wherein each type of micropellets comprisesinfluenza antigens or antigenic preparations from a different seasonal,pre-pandemic or pandemic influenza virus strain than the other type. 10.The process as claimed in claim 1, further comprising the addition of anaqueous solution comprising one or more adjuvants and, optionally, astabilizer to the liquid bulk antigen composition.
 11. The process asclaimed in claim 1, further comprising, a) diluting a separate liquidbulk adjuvant solution or emulsion comprising one or more adjuvants withan aqueous solution comprising a stabilizer, b) prilling the dilutedadjuvant solution or emulsion to form regular droplets of the adjuvanthaving a diameter of approximately from about 200 μm to about 1500 μm,c) subjecting the regular droplets of the adjuvant to freezing to formfrozen regular spherical micropellets or particles of the adjuvant, d)drying the frozen regular spherical micropellets or particles of theadjuvant to form dry regular spherical micropellets or particles of theadjuvant having a diameter from about 200 μm to about 1500 μm, e) andblending and filling the dry regular spherical micropellets or particlesof the adjuvant into a vial or other container together with the dryregular spherical micropellets of the vaccine.
 12. The process asclaimed in claim 10, wherein the adjuvant is selected from the groupconsisting of liposomes, lipid or detergent micelles or other lipidicparticles, polymer nanoparticles or microparticles, soluble polymers,protein particles, mineral gels, mineral micro- or nanoparticles,polymer/aluminum nanohybrids, oil in water or water in oil emulsions,saponin extracts, bacterial cell wall extracts, stimulators of innateimmunity receptors, natural or synthetic NOD agonists and natural orsynthetic RIG agonists.
 13. The process as claimed in claim 1, whereinthe influenza virus strains are selected from the group consisting ofH5N1, H9N2, H7N7, H2N2, H7N1 and H1N1.
 14. The process as claimed inclaim 1, wherein the influenza virus strains are selected from seasonalinfluenza virus strains.
 15. The process as claimed in claim 1, whereinthe influenza antigen is in a form of purified whole influenza virus,inactivated influenza virus, or sub-unit components of influenza virus.16. The process as claimed in claim 15, wherein the inactivatedinfluenza virus is a split influenza virus.
 17. The process as claimedin claim 1, wherein the influenza antigen is derived from cell culture.18. The process as claimed in claim 1, wherein the influenza antigen isproduced in embryonic eggs.
 19. A stabilized dry influenza vaccinecomposition in the form of dry regular spherical micropellets orparticles having a diameter from about 200 μm to about 1500 μm obtainedby the process according to claim
 1. 20. The composition as claimed inclaim 19 wherein each regular spherical micropellet or particlecomprises one or more influenza antigens from only one influenza virusstrain.
 21. The composition as claimed in claim 19 wherein each regularspherical micropellet or particle comprises one or more influenzaantigens from one or more different influenza virus strains.
 22. Thecomposition as claimed in claim 19 further comprising an adjuvant. 23.The composition of claim 22, wherein the adjuvant is contained inseparate dry regular spherical micropellets or particles.
 24. A processfor stabilizing an adjuvant-containing vaccine composition comprising,a) diluting a liquid bulk antigen composition comprising an antigen oran antigenic preparation with an aqueous solution comprising astabilizer and, optionally, one or more adjuvants to form a dilutedvaccine composition, b) prilling the diluted vaccine composition to formregular droplets of the vaccine having a diameter of approximately fromabout 200 μm to about 1500 μm, c) subjecting the regular droplets of thevaccine to freezing to form frozen regular spherical micropellets orparticles of the vaccine, d) drying the frozen regular sphericalmicropellets or particles of the vaccine to form dry regular sphericalmicropellets or particles of the vaccine having a diameter from about200 μm to about 1500 μm, and if the liquid bulk composition of (a) didnot comprise one or more adjuvants, e) diluting a separate liquid bulksolution or emulsion of one or more adjuvants with an aqueous solutioncomprising a stabilizer, f) prilling the diluted adjuvant solution oremulsion to form regular droplets of the adjuvant solution having adiameter of approximately from about 200 μm to about 1500 μm, g)subjecting the regular droplets to freezing to form frozen regularspherical micropellets or particles of the adjuvant solution, h) dryingthe frozen regular spherical micropellets or particles of the adjuvantsolution to form dry regular spherical micropellets or particles ofadjuvant composition having a diameter of from about 200 μm to about1500 μm, and i) blending and filling the dry regular sphericalmicropellets or particles of adjuvant composition into a vial or othercontainer together with the dry regular spherical micropellets orparticles of the vaccine.
 25. The process as claimed in claim 24,wherein the liquid bulk antigen composition further comprises one ormore additional antigens or antigenic preparations, each from adifferent pathogen or serotype of a pathogen, and wherein the dryregular spherical micropellets or particles of the vaccine each comprisetwo or more antigens or antigenic preparations from two or moredifferent pathogens or serotypes of a pathogen.
 26. The process asclaimed in claim 24, wherein the liquid bulk antigen compositioncomprises an antigen or antigenic preparation from a single pathogen orserotype of a pathogen, and wherein the dry regular sphericalmicropellets or particles of the vaccine each comprises antigens orantigenic preparations from the same pathogen or serotype of a pathogen.27. The process as claimed in claim 26, further comprising dosing,blending and filling into a vial or other container two or more types ofdry regular spherical micropellets or particles of the vaccine, whereineach type of micropellet or particle of the vaccine comprises antigensor antigenic preparations from two or more different pathogens orserotypes of a pathogen.
 28. The process as claimed in claim 24, whereinthe drying occurs by the method of lyophilization.
 29. The process asclaimed in claim 24, wherein the drying occurs by atmosphericfreeze-drying, fluidized bed drying, vacuum rotary drum drying, stirredfreeze-drying, vibrated freeze-drying, or microwave freeze-drying. 30.The process as claimed in claim 24, wherein the stabilizer is amonosaccharide, an oligosaccharide, a sugar alcohol, or a mixturethereof.
 31. A stabilized dry vaccine composition comprising one or moreantigens and adjuvant, which composition is in the form of dry regularspherical micropellets or particles having a diameter from about 200 μmto about 1500 μm obtained by the process according to claim
 24. 32. Thecomposition as claimed in claim 31 wherein each regular bead or particlecomprises only one type of antigen.
 33. The composition as claimed inclaim 31 wherein each regular bead or particle comprises one or moredifferent types of antigens.
 34. The composition of claim 31, whereinthe adjuvant is contained in separate dry regular spherical micropelletsor particles.
 35. The composition as claimed in claim 31 wherein theantigen or the antigens are selected from the group consisting of liveattenuated viruses, inactivated whole viruses, antigenic components ofviruses, whole bacteria, conjugated or non-conjugated bacterial protein,and conjugated or nonconjugated bacterial polysaccharide.
 36. A processfor the preparation a vaccine comprising reconstituting the compositionaccording to claim 19 or 31 in an aqueous solution.
 37. The process asclaimed in claim 36, wherein the aqueous solution comprises an adjuvant.38. A vaccine kit comprising a first container containing a stabilizeddry vaccine composition in the form of dry regular sphericalmicropellets or particles according to claim 19 or 31 and a secondcontainer containing an aqueous solution for the reconstitution of thevaccine.
 39. The kit as claimed in claim 38 wherein the aqueous solutioncomprises an adjuvant.
 40. The kit as claimed in claim 38 wherein itcomprises a third container containing a stabilized dry adjuvantcomposition in the form of dry regular spherical micropellets orparticles.
 41. A method of stockpiling a stable dry bulk of antigenswherein the antigen or the antigens is/are stabilized by a methodaccording to claim 1 or 24 and the resulting stabilized antigencomposition is reconstituted with an adequate solvent and optionallyformulated prior to liquid filling into vials or syringes after storagein the form of dry regular spherical micropellets or particles having adiameter from about 200 μm to about 1500 μm.
 42. The process as claimedin claim 10, wherein the adjuvant is natural or synthetic TLR agonists.43. The composition of claim 31, wherein the antigen or the antigens arelive attenuated viruses, inactivated whole viruses, or antigeniccomponents of viruses, and the viruses are selected from the groupconsisting of Polio, Influenza, Rotavirus, cytomegalo virus, andHepatitis A or B.
 44. The composition of claim 31, wherein the antigenor the antigens are whole bacteria or conjugated or non-conjugatedbacterial protein or conjugated or non-conjugated polysaccharideantigens, which are selected from the group consisting of meningococcalpolysaccharides, Tetanus, Diphtheria, cellular or acellular pertussis,Botulism, and Anthrax.